肿瘤单细胞的骨架蛋白定量及其力学特性高通量综合检测方法研究

肿瘤细胞存在异质性，即同一肿瘤样本中单个肿瘤细胞在生长、迁移、侵袭等方面存在差异。从细胞骨架层面来看，肿瘤细胞异质性主要表现为肿瘤细胞骨架的结构特征（骨架蛋白的数量、组成和分布）和功能特征（细胞的力学特性、迁移和侵袭）的差异。传统方法已经能够分别得到单个肿瘤细胞骨架的蛋白数量和力学特性这两个主要参数，但是存在测量通量低、绝对量化难、细胞骨架的结构和功能特征不能同时表征等缺陷，限制肿瘤细胞异质性的研究。在本项目中，申请人拟采用微流控、高速摄像、乳化液滴、荧光采集等技术，建立细胞高速通过横截面周期性变化的微流道的粘弹性等效力学仿真模型，研究高通量的获取细胞骨架力学特性的方法，探索量化乳化液滴中细胞骨架蛋白数量的荧光检测方法，解决细胞骨架的蛋白数量高通量绝对量化的难题，研究针对同一肿瘤细胞的细胞骨架的蛋白数量和力学特性高通量综合检测方法，为肿瘤细胞异质性的研究提供可靠的方法与途径。

Tumor heterogeneity is a key component of tumor cell biology, which is responsible for tumor variations in proliferation rates, invasion capabilities and drug treatment responses. From the perspective of cytoskeletons, tumor heterogeneity is demonstrated in different structures, assembles and distributions of cytoskeleton proteins, resulting in significant different capabilities of deformability, migration and invasion among individual tumor cells. However, due to limitations of technical tools capable of high-throughput characterizing cytoskeletons of single cells, protein differences and mechanical property variations of individual tumor cells within the same tumor samples remain elusive. In order to address this issue, in this proposal, we will develop a high-throughput measurement system to characterize cytoskeleton proteins and mechanical properties of single tumor cells by integrating microfluidic chips, high-speed imaging, emulsification droplets and fluorescence detection. More specifically, a mechanical model will be developed to understand the cellular travelling process in a periodic constriction channel, enabling high-throughput characterization of viscoelastic properties of single cells. Meanwhile, droplet microfluidic platforms will be proposed to confine single cells in individual droplets and characterize cytoskeleton proteins based on fluorescence staining. The developed system will quantify cytoskeleton proteins and mechanical properties of tumor cell lines and patient tumor samples, functioning as an effective tool for the study of tumor heterogeneity.